A basic question; I know should fixate and permeabilize cells before staining for intracellular markers, I wonder if there would be still possibility for staining the same markers which are expressed on cell surface. e.g. CD206(MR) which express both on surface and intracellular.
Hi Sahar,
You should stain for the surface markers first. Then fix and permeabilize and then stain for your intracellular markers. If you fix prior to staining for the surface markers you run the risk of denaturing the epitopes and the antibody may not bind. Hope this is helpful.
Good Luck!
Depends on the stability of the antigen/epitope; you have to evaluate that for your specific antigens of interest.
For a general protocol see e.g. http://www.cellsignal.com/contents/resources-protocols/protocol-for-combined-staining-of-intracellular-proteins-and-cell-surface-markers-in-blood/flow-alternative
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068519
Hi Tom,
Thanks for your answer. My main concern is intracellular antigens, don't they change configuration when we do fixation/permeabilization? And, when you stain the intracellular antigen, is there a chance that the the antibody binds to the same antigen with *surface expression*?
Fixation and permeabilization can certainly alter the conformation of a protein so it's no longer recognized by an antibody, but it could also be required for recognition in other cases. This is why antibodies differ so widely in the applications for which they are appropriate. I know from microscopy experiments that conditions necessary to permeabilize organelles can reduce plasma membrane staining. However, I think it's best to assume that an antibody used for intracellular flow will still recognize the same protein on the cell surface unless you show that it doesn't. The one suggestion I have is to stain for your protein on the cell surface using a fluorophore conjugated primary antibody, then wash, fix/perm, and stain for your protein again using the same antibody but with a different color fluorophore. As a control, I would do surface staining only and then measure the signal with or without a subsequent fix/perm step. You could also do the intracellular staining with a fluorescent primary antibody with or without prior surface staining with an unlabeled primary to block extracellular sites. These approaches might help you determine how much of your flow signal comes from the surface vs. intracellular pools. Of course, this all assumes your antibody recognizes an extracellular epitope; if it recognizes a cytoplasmic epitope, you would have to permeabilize even to detect plasma membrane proteins. Hope that helps!
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